Benzodiazepine derivatives, compositions containing them and their use in therapy

ABSTRACT

Compounds of Formula (I), and salts and prodrugs thereof wherein: R 1  is H, certain optionally substituted C 1-6  alkyl or C 3-7  cycloalkyl; R 2  represents a group (a) wherein X is O, S or NR 8  where R 8  is H or C 1-4  alkyl; one of Z and Y is C═O and the other is O, S or NR 9 , where R 9  is H or C 1-4  alkyl; R 3  is C 1-6  alkyl, halo or NR 6  R 7  ; R 4  is C 3-10  cycloalkyl; n is 0, 1, 2 or 3, and are CCK and/or gastrin antagonists, which compounds and compositions thereof are useful in therapy.

This application is a 35 U.S.C. §371 application of PCT/GB93/00535,filed Mar. 15, 1993.

This invention relates to benzodiazepine compounds which are useful asantagonists of cholecystokinin and gastrin receptors.

Cholecystokinins (CCK) and gastrin are structurally related peptideswhich exist in gastrointestinal tissue and in the central nervous system(see, V. Mutt, Gastrointestinal Hormones, G. B. J. Green, Ed., RavenPress, N.Y., p.169 and G. Nission, ibid. p.127).

Cholecystokinins include CCK-33, a neuropeptide of thirty-three aminoacids in its originally isolated form (see, Mutt and Jorpes, Biochem. J.125, 678 (1971)), its carboxylterminal octapeptide, CCK-8 (also anaturally-occurring neuropeptide and the minimum fully active sequence),and 39- and 12-amino acid forms. Gastrin occurs in 34-, 17- and 14-aminoacid forms, with the minimum active sequence being the C-terminaltetrapeptide, Trp-Met-Asp-Phe-NH₂, which is the common structuralelement shared by both CCK and gastrin.

CCKs are believed to be physiological satiety hormones, thereby possiblyplaying an important role in appetite regulation (G. P. Smith, Eatingand Its Disorders, A. J. Stunkard and E. Stellar, Eds, Raven Press, NewYork, 1984, p. 67), as well as stimulating colonic motility, gallbladder contraction, pancreatic enzyme secretion and inhibiting gastricemptying. They reportedly co-exist with dopamine in certain mid-brainneurons and thus may also play a role in the functioning of dopaminergicsystems in the brain, in addition to serving as neurotransmitters intheir own right (see A. J. Prange et al., "Peptides in the CentralNervous System" Ann. Repts. Med. Chem. 17, 31, 33 [1982] and referencescited therein; J. A. Williams, Biomed Res. 3 107 [1982]; and J. E.Morley, Life Sci. 30, 479 [1982]).

The primary role of gastrin, on the other hand, appears to bestimulation of the secretion of water and electrolytes from the stomachand, as such, is involved in control of gastric acid and pepsinsecretion. Other physiological effects of gastrin then include increasedmucosal blood flow and increased antral motility. Rat studies have shownthat gastrin has a positive trophic effect on the gastric mucosa, asevidenced by increased DNA, RNA and protein synthesis.

There are at least two subtypes of cholecystokinin receptors termedCCK-A and CCK-B (T. H. Moran et al., "Two brain cholecystokininreceptors: implications for behavioural actions", Brain Res., 362,175-79 [1986]). Both subtypes are found both in the periphery and in thecentral nervous system.

CCK and gastrin receptor antagonists have been disclosed for preventingand treating CCK-related and/or gastrin related disorders of thegastrointestinal (GI) and central nervous (CNS) systems of animals,especially mammals, and more especially those of humans. Just as thereis some overlap in the biological activities of CCK and gastrin,antagonists also tend to have affinity for both CCK-B receptors andgastrin receptors. Other antagonists have activity at the CCK-A subtype.

Selective CCK antagonists are themselves useful in treating CCK-relateddisorders of appetite regulatory systems of animals as well as inpotentiating and prolonging opiate-mediated analgesia [see P. L. Fariset al., Science 226, 1215 (1984)], thus having utility in the treatmentof pain. CCK-B and CCK-A antagonists have also been shown to have adirect analgesic effect [M. F. O'Neill et al., Brain Research, 534 287(1990)]. Selective CCK and gastrin antagonists are useful in themodulation of behaviour mediated by dopaminergic and serotonergicneuronal systems and thus have utility in the treatment of schizophreniaand depression (Rasmussen et. al., 1991, Eur. J. Pharmacol., 209,135-138; Woodruff et. al., 1991, Neuropeptides, 19, 45-46; Cervo et.al., 1988, Eur. J. Pharmacol., 158, 53-59), as a palliative forgastrointestinal neoplasms, and in the treatment and prevention ofgastrin-related disorders of the gastrointestinal system in humans andanimals, such as peptic ulcers, Zollinger-Ellison syndrome, antral Gcell hyperplasia and other conditions in which reduced gastrin activityis of therapeutic value, see e.g. U.S. Pat. No. 4,820,834. Certain CCKantagonists are useful anxiolytic agents and can be used in thetreatment of panic and anxiety disorders.

CCK has been reported to evoke the release of stress hormones such asadrenocorticotrophic hormone, β-endorphin, vasopressin and oxytocin, CCKmay function as a mediator of responses to stress and as part of thearousal system. CCK-A receptors are now known to be present in a numberof areas of the CNS and may be involved in modulating any of the above.

CCK may be involved in the regulation of stress and its relationshipwith drug abuse e.g. alleviation of the benzodiazepine withdrawalsyndrome (Singh et. al., 1992, Br. J. Pharmacol., 105, 8-10) andneuroadaptive processes.

Since CCK and gastrin also have trophic effects on certain tumours [K.Okyama, Hokkaido J. Med. Sci., 206-216 (1985)], antagonists of CCK andgastrin are useful in treating these tumours [see, R. D. Beauchamp etal., Ann. Surg., 202, 203 (1985)].

In the light of discussion in C. Xu et al., Peptides, 8, 1987, 769-772,CCK antagonists may also be effective in neuroprotection.

CCK receptor antagonists have been found to inhibit the contractileeffects of CCK on iris sphincter and ciliary muscles of monkey and humaneyes (Eur. J. Pharmacol., 211(2), 183-187; A. Bill et al., Acta Physiol.Scand., 138, 479-485 [1990]), thus having utility in inducing miosis fortherapeutic purposes.

A class of benzodiazepine antagonist compounds has been reported whichbinds selectively to brain CCK (CCK-B and CCK-A) and gastrin receptors[see M. Bock et al., J. Med Chem., 32, 13-16 (1989)].

European patent application no. 0 167 919 discloses benzodiazepine CCKand gastrin antagonists substituted in the 3-position by, inter alia, aphenyl urea and at the 5-position by an optionally substituted phenyl orC₁₋₄ alkyl group. There is no suggestion of the phenyl urea substitutionof the compounds of the present invention.

The present invention provides benzodiazepine compounds of formula (I)##STR2## wherein:

R¹ represents H, (CH₂)_(q) imidazolyl, (CH₂)_(q) tetrazolyl, (CH₂)_(q)triazolyl, (where q is 1, 2 or 3); C₁₋₆ alkyl optionally substituted byone or more groups selected from halo, hydroxy and NR⁶ R⁷ (where R⁶ andR⁷ each independently represents H or C₁₋₄ alkyl, or R⁶ and R⁷ togetherform a chain (CH₂)_(p) where p is 4 or 5); C₃₋₇ cycloalkyl;cyclopropylmethyl; CH₂ CO₂ R⁵ (where R⁵ is C₁₋₄ alkyl), CH₂ CONR⁶ R⁷ orCH₂ CH(OH)--W--(CH₂)₂ NR⁶ R⁷ where W is S or NH and R⁶ and R⁷ are aspreviously defined;

R² represents a group ##STR3## wherein: X represents O, S or NR⁸ whereR⁸ represents H or C₁₋₄ alkyl;

one of Z and Y is C═O and the other is O, S or NR⁹, where R⁹ representsH or C₁₋₄ alkyl;

R³ represents C₁₋₆ alkyl, NR⁶ R⁷, where R⁶ and R⁷ are as previouslydefined, or halo;

R⁴ represents C₃₋₁₀ cycloalkyl;

n is 0, 1, 2 or 3;

and salts and prodrugs thereof.

As used herein, the definition of each expression, when it occurs morethan once in any structure, is intended to be independent of itsdefinition elsewhere in the same structure.

It will be appreciated that formula (I) is intended to embrace allpossible isomers, including optical isomers, and mixtures thereof,including racemates.

The present invention includes within its scope prodrugs of thecompounds of formula (I) above. In general, such prodrugs will befunctional derivatives of the compounds of formula (I) which are readilyconvertible in vivo into the required compound of formula (I).Conventional procedures for the selection and preparation of suitableprodrug derivatives are described, for example, in "Design of Prodrugs",ed. H. Bungaard, Elsevier, 1985.

As used herein, alkyl means linear or branched chain alkyl. Examples ofsuitable alkyl groups include methyl, ethyl, isopropyl and isobutylgroups.

When R¹ represents cycloalkyl, examples of suitable cycloalkyl groupsinclude cyclopropyl, cyclopentyl and cyclohexyl groups, preferablycyclopropyl.

Halo includes fluoro, chloro, bromo and iodo. Preferably halo will befluoro or chloro.

A subgroup of compounds of the present invention is represented bycompounds of formula (I) wherein R³ represents C₁₋₆ alkyl or halo; R⁴represents C₃₋₇ cycloalkyl; and n is 0, 1 or 2.

Preferably R¹ is C₁₋₆ alkyl, more preferably C₁₋₄ alkyl, such as methyl,n-propyl or isobutyl.

Suitable examples of the substituent R² include ##STR4##

Preferably R² represents oxadiazolinone.

Preferably R² is in the 3- or 4-position of the phenyl ring, morepreferably the 3-position.

Suitable values for R³ include methyl, dimethylamino, chloro and bromo.

Preferably n is 0 or 1, more preferably 0.

Suitable values for R⁴ include cyclobutyl, cyclopentyl, cyclohexyl andcycloheptyl. Preferably R⁴ represents cyclohexyl.

A preferred sub-group of compounds according to the invention isrepresented by compounds of formula (IA), and salts and prodrugsthereof: ##STR5## wherein R⁴ is as defined for formula (I) above and R²⁰represents C₁₋₆ alkyl, preferably C₁₋₄ alkyl.

Preferably the salts of the compounds of formula (I) arepharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay be used for the preparation of pharmaceutically acceptable salts.The pharmaceutically acceptable salts of the compounds of formula (I)include the conventional non-toxic salts or the quaternary ammoniumsalts of the compounds from formula (I) formed, e.g., from non-toxicinorganic or organic salts. For example, such conventional non-toxicsalts include basic salts, e.g. sodium and potassium salts and thosederived from inorganic acids such as hydrochloric, hydrobromic,sulphuric, sulphamic, phosphoric, nitric and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, steric, lactic, malic, tartaric, citric, ascorbic, palmoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,sulphanilic, 2-acetoxy benzoic, fumaric, toluenesulphonic,methanesulphonic, ethane disulphonic, oxalic and isothionic.

The pharmaceutically acceptable salts of the present invention can besynthesized from the compound of formula (I) which contain a basic oracidic moiety by conventional chemical methods. Generally, the salts areprepared by reacting the free base or acid with stoichiometric amountsor with an excess of the desired salt-forming inorganic or organic acidor base in a suitable solvent or combination of solvents.

For example, an acid of formula (I) may be reacted with an appropriateamount of a base, such as an alkali or alkaline earth metal hydroxidee.g. sodium, potassium, lithium, calcium, or magnesium, or an organicbase such as an amine, e.g. dibenzylethylenediamine, trimethylamine,piperidine, pyrrolidine, benzylamine, and the like, or a quaternaryammonium hydroxide such as tetramethylammonium hydroxide.

The compounds of formula (I) antagonise CCK and/or gastrin and areuseful for the treatment and prevention of disorders including centralnervous system disorders wherein CCK and/or gastrin may be involved.Examples of such disease states include gastrointestinal diseases,including gastrointestinal ulcers, such as peptic and duodenal ulcers,irritable bowel syndrome, gastroesophagenal reflux disease or excesspancreatic or gastrin secretion, acute pancreatitis, or motilitydisorders; central nervous system disorders, including central nervoussystem disorders caused by CCK interaction with dopamine, serotonin andother monoamine neurotransmitters, such as neuroleptic disorders,tardive dyskinesia, Parkinson's disease, psychosis or Gilles de laTourette syndrome; depression, such as depression resulting from organicdisease, secondary to stress associated with personal loss, oridiopathic depression; schizophrenia; disorders of appetite regulatorysystems; Zollinger-Ellison syndrome, antral and cell hyperplasia, orpain.

The compounds of formula (I) are particularly useful in the treatment orprevention of neurological disorders involving anxiety disorders andpanic disorders, wherein CCK and/or gastrin is involved. Examples ofsuch disorders include panic disorders, anxiety disorders, panicsyndrome, anticipatory anxiety, phobic anxiety, panic anxiety, chronicanxiety and endogenous anxiety.

The compounds of formula (I) are also useful for directly inducinganalgesia, opiate or non-opiate mediated, as well as anesthesia or lossof the sensation of pain.

The compounds of formula (I) may further be useful for preventing ortreating the withdrawal response produced by chronic treatment or abuseof drugs or alcohol. Such drugs include, but are not limited tobenzodiazepines, cocaine, alcohol and nicotine.

The compounds of formula (I) may further be useful in the treatment ofstress and its relationship with drug abuse.

The compounds of formula (I) may further be useful in the treatment ofoncologic disorders wherein CCK may be involved. Examples of suchoncologic disorders include small cell adenocarcinomas and primarytumours of the central nervous system glial and neuronal cells. Examplesof such adenocarcinomas and tumours include, but are not limited to,tumours of the lower oesophagus, stomach, intestine, colon and lung,including small cell lung carcinoma.

The compounds of formula (I) may also be useful as neuroprotectiveagents, for example, in the treatment and/or prevention ofneurodegenerative disorders arising as a consequence of suchpathological conditions as stroke, hypoglycaemia, cerebral palsy,transient cerebral ischaemic attack, cerebral ischaemia during cardiacpulmonary surgery or cardiac arrest, perinatal asphyxia, epilepsy,Huntington's chorea, Alzheimer's disease, Amyotrophic Lateral Sclerosis,Parkinson's disease, Olivo-ponto-cerebellar atrophy, anoxia such as fromdrowning, spinal cord and head injury, and poisoning by neurotoxins,including environmental neurotoxins.

The compounds of formula (I) may further be used to induce miosis fortherapeutic purposes after certain types of examination and intraocularsurgery. An example of intraocular surgery would include cateractsurgery with implantation of an artificial lens. The CCK antagonistcompounds of this invention can be used to prevent miosis occuring inassociation with iritis, ureitis and trauma.

The present invention therefore provides a compound of formula (I) or asalt or prodrug thereof for use in the preparation of a medicament.

The present invention also provides a compound of formula (I) or a saltor prodrug thereof for use in therapy.

In a further or alternative embodiment the present invention provides amethod for the treatment or prevention of a physiological disorderinvolving CCK and/or gastrin which method comprises administration to apatient in need thereof of a CCK and/or gastrin antagonising amount of acompound of formula (I).

When a compound according to formula (I) is used as an antagonist of CCKor gastrin in a human subject, the daily dosage will normally bedetermined by the prescibing physician with the dosage generally varyingaccording to the age, weight, and response of the individual patient, aswell as the severity of the patient's symptoms. However, in mostinstances, an effective daily dosage wll be in the range from about0.005 mg/kg to about 100 mg/kg of body weight, and preferably, of from0.05 mg/kg to about 50 mg/kg, such as from about 0.5 mg/kg to about 20mg/kg of body weight, administered in single or divided doses. In somecases, however, it may be necessary to use dosages outside these limits.For example, animal experiments have indicated that doses as low as 1 ngmay be effective.

In effective treatment of panic syndrome, panic disorder, anxietydisorder and the like, preferably about 0.05 mg/kg to about 0.5 mg/kg ofCCK antagonist may be administered orally (p.o.), administered in singleor divided doses per day (b.i.d.). Other routes of administration arealso suitable.

For directly inducing analgesia, anaesthesia or loss of pain sensation,the effective dosage preferably ranges from about 100 ng/kg to about 1mg/kg by systemic administration. Oral administration is an alternativeroute, as well as others.

In the treatment or irritable bowel syndrome, preferably about 0.1 to 10mg/kg of CCK antagonist is administered orally (p.o.), administered insingle or divided doses per day (b.i.d.). Other routes of administrationare also suitable.

The use of a gastrin antagonist as a tumour palliative forgastrointestinal neoplasma with gastrin receptors, as a modulator ofcentral nervous activity, treatment of Zollinger-Ellison syndrome, or inthe treatment of peptic ulcer disease, an effective dosage of preferablyabout 0.1 to about 10 mg/kg administered one-to-four times daily isindicated.

For use as neuroprotective agents the effective dosage preferably rangesfrom about 0.5 mg/kg to about 20 mg/kg.

Because these compounds antagonise the function of CCK in animals, theymay also be used as feed additives to increase the food intake ofanimals in daily dosage of preferably about 0.05 mg/kg to about 50 mg/kgof body weight.

The compounds of formula (I) may be prepared by processes analogous tothose described in European Patent Specification No. 0284256. Forexample, a compound of formula (I) may be prepared by reacting anintermediate of formula (II) with an intermediate of formula (III)##STR6## wherein R¹, R², R³, R⁴ and n are as defined for formula (I),and one of R³⁰ and R³¹ represents NH₂ and the other of R³⁰ and R³¹represents --N═C═O.

The reaction is preferably conducted in a suitable organic solvent, suchas an ether, for example, tetrahydrofuran, at room temperature.

Intermediates of formula (II) wherein R³⁰ represents NH₂ (IIA) may beprepared from compounds of formula (VI) ##STR7## wherein R³, R⁴ and nare as defined for formula (I) and Z is a protecting group; by reactionwith a reagent suitable to introduce the group R¹, for example a halideof formula R¹ Hal where Hal represents halo such as bromo or iodo, inthe presence of a base, such as an alkali metal hydride or an alkalineearth metal carbonate, for example sodium hydride or caesium carbonate;or a suitable dialkyl acetal of dimethyl formamide in a suitable organicsolvent, e.g. toluene followed by deprotection.

Compounds of formula (VI) may be prepared from compounds of formula(VII) ##STR8## wherein R³, R⁴ and n are as defined for formula (I) andR¹¹ is H, by a reaction sequence comprising:

(i) reaction with a compound of formula (VIII) ##STR9## wherein Z is asdefined above, in the presence of a base, such as a tertiary amine, forexample triethylamine or N-methyl morpholine, and a coupling reagent.Any of the coupling reagents commonly used in peptide synthesis aresuitable, for example, 1,3-dicyclohexylcarbodiimide (DCC), isobutylchloroformate or, preferably, bis(2-oxo-3-oxazolidinyl)phosphinicchloride (BOP-Cl);

(ii) Treatment with gaseous ammonia, preferably in the presence of amercury containing catalyst, such as mercury(II) chloride. The reactionis conveniently effected in a suitable organic solvent, such as anether, for example, tetrahydrofuran;

(iii) Treatment with an organic acid, for example acetic or propionicacid, optionally in the presence of an ammonium salt, for exampleammonium acetate.

Compounds of formula (VII) wherein R¹¹ is H may be prepared fromcorresponding compounds of formula (VII) wherein R¹¹ is COCH₃ bytreatment with a mineral acid, for example hydrochloric acid, or basehydrolysis, for example, using aqueous sodium hydroxide. The reaction isconveniently affected in refluxing methanol.

Alternatively, compounds of formula (VII) wherein R¹¹ is H may beprepared by reaction of a compound of formula (IX) ##STR10## wherein R³and n are as previously defined, with a Grignard reagent of formula R⁴MgHal wherein R⁴ is as previously defined and Hal is halo such aschloro, bromo or iodo.

Compounds of formula (IX) are commercially available or may be preparedfrom commercially available compounds by conventional methods.

Compounds of formula (VII) wherein R¹¹ is COCH₃ may be prepared fromcompounds of formula (X) ##STR11## wherein R³ and n are defined as forformula (I), by reaction with a Grignard reagent of formula R⁴ MgHalwherein Hal is halo such as chloro, bromo or iodo.

Compounds of formula (X) may be prepared by known methods, e.g. see D.A. Walsh, Synthesis, 677, (1980).

Where the above-described process for the preparation of the compoundsaccording to the invention gives rise to mixtures of stereoisomers theseisomers may, if desired, be separated, suitably by conventionaltechniques such as preparative chromatography.

The novel compounds may be prepared in racemic form, or individualenantiomers may be prepared either by enantiospecific synthesis or byresolution. The novel compounds may, for example, be resolved into theircomponent enantiomers by standard techniques, such as the formation ofdiastereomeric pairs by salt formation with an optically active acid,such as (-)-di-p-toluoyl-L-tartaric acid and/or(+)-di-p-toluoyl-D-tartaric acid followed by fractional crystallizationand regeneration of the free base. The novel compounds may also beresolved by formation of diastereomeric esters or amides, followed bychromatographic separation and removal of the chiral auxiliary.Alternatively, enantiomers of the novel compounds may be separated byHPLC using a chiral column.

During any of the above synthetic sequences it may be necessary and/ordesirable to protect sensitive or reactive groups on any of themolecules concerned. This may be achieved by means of conventionalprotecting groups, such as those described in Protective Groups inOrganic Chemistry, ed. J. F. W. McOmie, Plenum Press, 1973; and T. W.Greene and P. G. M. Wutts, Protective Groups in Organic Synthesis, JohnWiley & Sons, 1991. The protecting groups may be removed at a convenientsubsequent stage using methods known from the art.

The following examples are provided to assist in a further understandingof the invention. Particular materials employed, species and conditionsare intended to be further illustrative of the invention and notlimitative of the scope thereof.

EXAMPLE 1 N-[3(R)-5-Cyclohexyl-2,3-dihydro-1-methyl-2-oxo-1H-1,4-benzodiazenin-3-yl]N'[3-(5-oxo-4H-1,2,4-oxadiazolin-3-l)phenyl] urea Intermediate 1(+)-3(R)-Amino-5-cyclohexyl-1,3-dihydro-1-methyl-2H-1,4-benzodiazenin-2-one

Step 1: (2-Acetamidophenyl) cyclohexyl methanone

Cyclohexylmagnesium bromide (240 ml of a 2M solution in ether, 0.48 mol)in ether (200 ml) was added dropwise to a solution of2-methyl-4H-3,1-benzoxazin-4-one (100 g, 0.62 mol) in ether (1100ml) at-10° C. over 2 h. The mixture was stirred at this temperature for 2 h,then at ambient temperature for 30 min. After cooling to -10° C. thesuspension was treated with 2M HCl (600 ml), keeping the temperaturebelow 0° C. After stirring for 15 min the layers were separated, and theethereal layer washed sequentially with water (500 ml), 5% sodiumhydroxide solution (2×500 ml) and finally water (2×500 ml). The organiclayer was separated, dried (MgSO₄), evaporated in vacuo andchromatographed on silica using petrol:ethyl acetate (2:1). to give(2-acetamidophenyl) cyclohexyl methanone (28 g, 24%) as a pale yellowsolid. mp 66° C. ¹ H NMR (CDCL₃, 360 MHz)₋₋ 1.25-1.89 (10H, m), 2.23(3H, s), 3.33 (1H, m), 7.13 (1H, dt, J=6 and 1 Hz), 7.53 (1H, dt, J=6and 1 Hz), 7.92 (1H, d, J=6 Hz), 8.76 (1H, d, J=6 Hz), 11.73 (1H, brs).

Step 2: (2-Aminophenyl) cyclohexyl methanone

A solution of (2-acetamidophenyl) cyclohexyl methanone (0.53 g, 2.16mmol) in methanol (5 ml) and concentrated hydrochloric add (15 ml) washeated at 80° C. for 1 h. After this time the solution was cooled toambient temperature and the solvents removed in vacuo. The residue wasdissolved in water (10 ml) and basified with 4N sodium hydroxidesolution (20 ml). The mixture was then extracted into ethyl acetate(4×20 ml) and the organic layers combined and dried (MgSO₄). The solventwas evaporated and the residue chromatographed on silica gel, usingpetrol:ethyl acetate (2:1), to afford the amine (0.40 g, 91%) as a whitesolid. mp 73°-75° C. ¹ H NMR (360 MHz, CDCL₃)₋₋ 1.23-2.09 (10H, m), 3.27(1H, m), 6.29 (2H, brs), 6.64 (2H, m), 7.25 (1H, dt, J=6 and 1 Hz), 7.76(1H, dd, J -7 and 1 Hz).

An alternative procedure could be used for preparation of(2-aminophenyl)cyclohexyl methanone: To a cooled (0° C.) and stirredsolution of 2-aminobenzonitrile (59.5 g, 0.5 mmol) in anhydrous diethylether (210 ml) was added dropwise cyclohexylmagnesium chloride (2M indiethyl ether, 700 ml) at such a rate as to maintain the temperaturebelow 25° C. After a further 18 h stirring at room temperature, themixture was cooled to -60° C. and treated dropwise (CAUTION! highlyexothermic reaction) with 5N hydrochloric add (600 ml). The mixture wasthen allowed to warm to room temperature, diluted with additional 5Nhydrochloric add (500 ml) and the ethereal layer was separated. Theacidic aqueous solution was basified to pH 4-5 with solid potassiumhydroxide and then extracted with ethyl acetate (3×700 ml). The etherealand ethyl acetate solutions were combined, washed with brine (1000 ml),dried (MgSO₄) and concentrated under vacuum to give the title compound(97 g, 94%) as a pale yellow solid.

Step 3:3(R,S)-[(Benzyloxycarbonyl)amino]-5-cyclohexyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one

α-(Isopropylthio)-N-(benzyloxycarbonyl)glycine (30 g, 0.11 mol) wasdissolved in dichloromethane (1000 ml) and cooled to 0° C. The stirredsolution was then treated with N-methyl morpholine (11.5 ml, 0.11 mol)followed by isobutyl chloroformate (13.7 ml, 0.11 mol). The resultingreaction mixture was stirred for a further 15 min at 0° C., then heatedto reflux. The refluxing reaction mixture was treated dropwise, over 20min, with a solution of (2-aminophenyl) cyclohexyl methanone (20.5 g,0.1 mmol) in dichloromethane (140 ml). After addition was complete thereaction was heated at reflux for a further 4 h. The mixture was thenwashed in succession with 10% citric acid solution (2×500 ml), saturatedsodium bicarbonate solution (2×500 ml) and brine (500 ml). The dried(MgSO₄) organic phase was evaporated to afford the crude product as apale orange solid, which was used without further purification.

The crude (isopropylthio)glycinamide was dissolved in anhydroustetrahydrofuran (800 ml) and cooled to 0° C. Ammonia gas was bubbledthrough the stirred solution for 30 min before adding mercuric chloride(33 g, 0.12 mol) in one portion. Ammonia was continually bubbled throughthe solution for a further 5 hours, then the suspended solids werefiltered off. The solvent was evaporated in vacuo to leave an oil, whichwas used without further purification.

The crude α-aminoglycinamide was dissolved in glacial acetic acid (500ml) and treated with ammonium acetate (36.2 g, 0.47 mol). The resultingreaction mixture was stirred at room temperature overnight, beforeremoving the solvent in vacuo. The residue was partitioned between ethylacetate (300 ml) and 1N sodium hydroxide solution (300 ml). The organicphase was separated, dried (MgSO₄) and evaporated. The residue waschromatographed on silica, using 2:1 petrol:ethyl acetate as the eluant,to afford3(R,S)-[(benzyloxycarbonyl)amino]-5-cyclohexyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one(25 g, 64%) as a white solid. mp 164-166° C. ¹ H NMR (360 MHz, CDCL₃)δ1.07-2.04 (10H, m), 2.77 (1H, m), 5.12 (3H, m), 6.44 (1H, d, J=8 Hz),7.08 (1H, d, J=8 Hz), 7.23-7.36 (6H, m), 7.46 (1H, t, J=7 Hz), 7.59 (1H,d, J=8 Hz), 8.60 (1H, brs).

Step 4:3(R,S)-[(Benzyloxycarbonyl)amino]-5-cydohexyl-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one

A solution of3(R,S)-[(benzyloxycarbonyl)amino]-5-cyclohexyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one(1.1 g, 2.8 mmol) in dimethylformamide (13 ml), under an atmosphere ofnitrogen, was treated with sodium hydride (117 mg of a 55-60% dispersionin mineral oil, 2.8 mmol) in one portion, at -10° C. After 30 min at-10° C., iodomethane (174 μl, 2.8 mmol) was added in one portion and thesolution allowed to reach 0° C. over 1 h. The solvent was then removedin vacuo and the crude residue partitioned between water (100 ml) anddichloromethane (100 ml). The organic phase was separated and theaqueous phase extracted with dichloromethane (2×100 ml). The combinedorganic layers were washed with brine, dried (MgSO₄) and evaporated. Theresidue was chromatographed on silica, using 1:1 petrol:ethyl acetate asthe eluant, to afford the title compound (0.75 g, 66%) as a white solid.mp 205-207° C. ¹ H NMR (360 MHz, CDCl₃) δ1.03-2.04 (10H, m), 2.76 (1H,m), 3.36 (3H, s), 5.10 (3H, m), 6.52 (1H, d, J=8 Hz), 7.25-7.55 (9H, m).

Step 5:3(R,S)-Amino-5-cyclohexyl-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one

A mixture of3(R,S)-[(benzyloxycarbonyl)amino]-5-cyclohexyl-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one(3.0 g, 7.4 mmol) and hydrobromic acid (45% in acetic acid, 6.2 ml) wasstirred for 1 h at room temperature under an atmosphere of nitrogen. Themixture was then diluted with cold anhydrous diethyl ether (40 ml) andit was stirred at 0° C. for 45 min. The white precipitate was collectedby filtration, washed with cold diethyl ether (4×30 ml) and thendissolved in a mixture of water (30 ml) and aqueous sodium hydroxide(2M, 15 ml). The basic aqueous phase was extracted with ethyl acetate(3×70 ml) and the combined organic layers were washed with brine (30ml), dried (Na₂ SO₄) and concentrated. The residue was chromatographedon silica gel using 94:6, dichloromethane: methanol as the eluant, toafford the title compound (1.6 g, 80%) as pale pink solid. mp 133-136°C. ¹ H NMR (360 MHz, CDCl₃)δ1.02-1.40 (4H, m), 1.47-1.56 (1H, m),1.61-1.74 (3H, m), 1.84-1.91 (1H, m), 1.96-2.06 (1H, m), 2.17 (2H, brs), 2.70-2.80 (1H, m), 3.39 (3H, s), 4.29 (1H, s), 7.20-7.27 (2H, m),7.44-7.54 (2H, m).

Step 6:3(R,S)-[2(R)-(tert-Butyloxycarbonyl)amino-3-phenylpropionylamino]-5-cyclohexyl-1,3-dihydro-1-methyl-2H-1,4-benzodiazenin-2-one

To a solution of3(R,S)-amino-5-cyclohexyl-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one(4 g, 14.8 mmol) in anhydrous dimethylformamide (35 ml), under anatmosphere of nitrogen, was added in succession Boc-D-phenylalanine(4.11 g, 15.4 mmol), 1-hydroxybenzotriazole trihydrate (2.09 g, 15.4mmol) and 1-ethyl-3-[3-(dimethylamino) propyl]carbodiimide hydrochloride(2.97 g, 15.4 mmol). Triethylamine (2.16 ml, 15.4 mmol) was then addedand the resulting suspension was stirred at ambient temperature for 20min. The solvent was removed under reduced pressure and the residue waspartitioned between ethyl acetate (50 ml) and 10% citric acid solution(50 ml). The organic phase was separated and the aqueous phase extractedwith ethyl acetate (3×50 ml). The combined organic phases were washedwith 10% sodium hydroxide solution (50 ml), water (50 ml) and brine (50ml), dried (MgSO₄) and evaporated in vacuo. The residue waschromatographed on silica, using 1:1 petrol:ethyl acetate as the eluant,to afford the product (7.26, 95%) as a pale yellow solid. mp 95-98° C. ¹H NMR (360 MHz, CDCl₃) δ0.99-1.11 (1H, m), 1.16-1.72 (7H, m), 1,40 (9H,s), 1.83-1.92 (1H, m), 1.98-2.06 (1H, m), 2.73-2.83 (1H, m), 3.10-3.24(2H, m), 3.38 (3H, s), 4.53 (1H, brs), 4.98 (1H, brs), 5.28-5.34 (2H,m), 7.19-7.32 (7H, m), 7.49-7.58 (2H, m).

Step 7:(+)-3(R)-(2(R)-Amino-3-phenylpropionylamino)-5-cyclohexyl-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one

3(R,S)-[2(R)-(tert-Butyloxycarbonyl)amino-3-phenylpropionylamino]-5-cyclohexyl-1,3-dihydro-1-methyl-2H-1,4-benzodiaze pin-2-one(4.7 g, 9.1 mmol) was dissolved in ethyl acetate (20 ml) and cooled to0° C. This solution was then saturated with hydrogen chloride gas. After1 h 30 min, the resulting precipitate (which was shown to be theundesired diastereoisomer, R_(f) =0.04 ethyl acetate), was removed byfiltration and the filtrate evaporated. The solid residue waspartitioned between ethyl acetate (25m1) and 10% sodium carbonatesolution (20 ml). The 25 organic phase was separated and the aqueousextracted with ethyl acetate (2×25 ml). The combined organic phases weredried (Na₂ SO₄) and evaporated in vacuo. The residue was chromatographedon silica using a gradient elution of 0-20% methanol in ethyl acetate toafford the title compound (1.66 g, 44%, R_(f) =0.13 ethyl acetate) as apale yellow solid. mp 100-103° C. ¹ H NMR (360 MHz, CDCl₃) δ1.00-1,39(4H, m), 1.50-1.72 (4H, m), 1.84-1.92 (1H, m), 2.00-2.07 (1H, m),2.72-2.84 (1H, m), 2.79 (1H, dd, J=13.8 and 9.8 Hz), 3.28 (1H, dd,J=13.8 and 4.0 Hz), 3.40 (3H, s), 3.69 (1H, dd, J=9.8 and 4.1 Hz), 5.36(1H, d, J=8.3 Hz), 7.21-7.36 (7H, m), 7.47-7.58 (2H, m), 8.66 (1H, d,J=8.3 Hz). [α]²³ _(D) +32.7°(c=0.58,CH₃ OH).

The undesired diastereoisomer (Rf 0.04, ethyl acetate) could beepimerised to3(R,S)-(2(R)-amino-3-phenylpropionylamino)-5-cyclohexyl-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-oneusing the following procedure:

The undesired diastereoisomer (Rf 0.04, ethyl acetate) (16.8 g, 0.044mol) was dissolved in anhydrous ether (200 ml), andpotassiuxn-tert-butoxide (0.68 g, 6.1 mmol) was added. The mixture wasstirred at room temperature for 1 h, then more potassium-tert-butoxide(0.68 g, 6.1 mmol) was added and the mixture heated at reflux for 5 h.The mixture was then cooled to ambient temperature, the solvent removedunder vacuum, and the residue partitioned between ethyl acetate (200ml)and water (200 ml). The organic layer was separated, dried (MgSO₄),filtered and evaporated in vacuo to afford the epimerised material.

Step 8:(+)-N-[1(R)-2-[(3(R)-5-Cyclohexyl-2,3-dihydro-1-methyl-2-oxo-1H-1,4-benzodiazepin-3-yl)amino]-2-oxo-1-(phenylmethyl)ethyl]N'-phenyl thiourea

A solution of (+)-3(R)-(2(R)-amino-3-phenylpropionylamino)-5-cyclohexyl-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (1.6 g, 3.83 mmol) in anhydrous dichloromethane(10 ml) was treated with phenyl isothiocyanate (0.5 ml, 4.21 mmol), andthen heated on the steam bath for 30 min. The solvent was evaporated invacuo and the residue was chromatographed on silica with 1:1, ethylacetate:petrol to afford the product (2.1 g, 100%) as a pale yellowsolid. mp 129-132° C. ¹ H NMR (360 MHz, CDCl₃) δ0.95-1.07 (1H, m),1.15-1.37 (3H, m), 1.45-1.69 (4H, m), 1.81-1.88 (1H, m), 1.93-2.00 (1H,m), 2.70-2.80 (1H, m), 3.24-3.41 (2H, m), 3.38 (3H, s), 5.23 (1H, d,J=7.3 Hz), 5.31-5.40 (1H, m), 6.67 (1H, 7.0 Hz), 6.87-7.02 (2H, m),7.20-7.35 (9H, m), 7.46-7.52 (2H m), 7.65 (1H, s). [α]²⁵ _(D)+27.3°(c=0.31, CH₂ Cl₂).

Step 9:(+)-3(R)-Amino-5-cyclohexyl-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one

N-[1(R)-2-[(3(R)-5-Cyclohexyl-2,3-dihydro-1-methyl-2-oxo-1H-1,4-benzodiazepin-3-yl)amino]-2-oxo-1-(phenylmethyl)ethyl]N'-phenylthiourea (4.5 g, 8.1 mmol ) was dissolved in trifiuoroacetic acid (25ml) and stirred at ambient temperature for 30 min. The trifiuoroaceticacid was removed under reduced pressure and the residue azeotroped withdichloromethane (2×20 ml) and toluene (2×20 ml). The residue waschromatographed on silica gel using 90:10:0.1:0.1, dichloromethane:methanol:acetic add:water as the eluant to afford an orange gum. Thiswas dissolved in ethyl acetate (150 ml), cooled to 0° C., and treatedwith 10% sodium carbonate solution (15 ml). After diluting with water(25 ml) and stirring for 1 min, the organic layer was separated and theaqueous re-extracted with ethyl acetate (2×50 ml). The combined organicswere dried (Na₂ SO₄) and evaporated in vacuo to afford the titlecompound (1.56 g, 71%) as a solid with 99% e.e. mp 133-136° C. ¹ H NMR(360 MHz, CDCl₃) δ1.01-1.39 (4H, m), 1.50-1.54 (1H, m), 1.60-1.70 (3H,m), 1.84-1.92 (1H, m), 1.96-2.04 (1H, m), 2.36 (2H, brs), 2.70-2.80 (1H,m), 3.41 (3H, s), 4.32 (1H, s), 7.22-7.28 (2H, m), 7.46-7.58 (2H, m).[α]²³ _(D) +33.2°(c=0.66,CH₃ OH).

Intermediate 2 1-(5-Oxo-4H -1,2,4-oxadiazolin-3-yl)-3- aminobenzenehydrochloride salt

Step 1: 1-Cyano-3-tert-butyloxycarbonylaminobenzene

To a solution of 3-minobenzonitrile (47 g, 0.40 mol) in dichloromethane(210 ml) was added a solution of di-tert-butyldicarbonate (132 g, 0.60mmol) in dichloromethane (150 ml). The mixture was heated at reflux for4 days then the mixture cooled to room temperature, and stirred with 10%citric acid solution (200 ml). The organic layer was separated and theaqueous phase extracted with dichloromethane (3×100 ml). The organiclayers were combined, washed with brine (100 ml) then separated. Afterdrying (Na₂ SO₄) the solvent was evaporated to afford a beige solid.This was triturated in petroleum ether (60/80) and the desired productcollected as a white solid (65.9 g, 7.5%). mp 123-126° C. ¹ H NMR (360MHz, CDCl₃)δ1.52 (9H, s), 6.50 (1H, brs), 7.29 (1H, d, J=8 Hz), 7.36(1H, dd, J=8 and 8 Hz), 7.51 (1H, d, J=8 Hz), 7.76 (1H, s).

Step 2: 3-tert-Butyloxycarbonylaminobenzamide oxime

Hydroxylamine hydrochloride (3.16 g, 0.045 mol) was added to a stirredsolution of sodium ethoxide in ethanol [prepared by dissolving 1.17 g ofsodium in 70 ml of ethanol]. The mixture was stirred at room temperaturefor 15 min then 1-cyano-3-tert-butyloxycarbonylaminobenzene (3.00 g,0.014 mol) was added. The mixture was heated at 50° C. overnight thencooled to ambient temperature. The mixture was then filtered and thefiltrate evaporated in vacuo. The residue was partitioned between ethylacetate (50 ml) and water (50 ml). The organic phase was separated andthe aqueous phase extracted with ethyl acetate (3×20 ml). The organiclayers were combined, washed with brine (50 ml) then dried (Na₂ SO₄).The solvent was evaporated to leave a pink fo.m. The foam was left at 0°C. overnight, then triturated with 1:1 petrol:ethyl acetate. The titlecompound was collected as a white solid (2.52 g, 73%). ¹ H NMR (360 MHz,D₆ -DMSO)δ1.47 (9H, s), 5.69 (2H, brs), 7.24 (2H, m), 7.41 (1H, brd,J=6.5 Hz), 7.82 (1H, s), 9.35 (1H, s), 9.58 (1H, s). MS (CI, NH₃) 252(M+1).

Step 3:1-(5-Oxo-4H-1,2,4-oxadiazolin-3-yl)-3-tert-butyloxycarbonlylaminobenzene

To a stirred solution of the amide oxime (0.68 g, 2.7 mmol) intetrahydrofuran (10 ml) was added 1,1-carbonyldiimidazole (0.53 g, 3.3mmol). The solution was stirred at room temperature overnight, afterwhich time a white solid had precipitated from solution. The mixture wasthen heated at reflux for 5 h then allowed to cool to ambienttemperature. The solvent was removed in vacuo and the residuepartitioned between dichloromethane (50 ml) and 10% citric add solution(50 ml). The organic layer precipitated a white solid which wascollected by filtration. The solid was identified as the desiredoxadiazolinone (0.53 g, 71%). mp 186-189° C. ¹ H NMR (360 MHz, D₆-DMSO)δ1.49 (9H, s), 7.36 (1H, d, J=9 Hz), 7.44 (1H, dd, J=8 and 8 Hz),7.58 (1H, d, J=9 Hz), 8.07 (1H, s), 9.64 (1H, s), 13.00 (1H, brs).

Step 4: 1-(5-Oxo-4H-1,2,4-oxadiazolin-3-yl)-3-aminobenzene hydrochloridesalt

1-(5-Oxo-4H-1,2,4-oxadiazolin-3-yl)-3-tert-butyloxycarbony laminobenzene(0.8 g, 2.9 mmol) was dissolved in ethyl acetate (40 ml) and cooled to0° C. Hydrogen chloride gas was then bubbled through the stirredsolution for 10 min. Nitrogen was bubbled through the mixture for 10 minthen the solvent evaporated in vacuo. The residue was azeotroped withtoluene (2×20 ml) then triturated with ether. The title compound (0.59g, 95%) was isolated as a white solid. mp 238-242° C. (dec.). 1H NMR(360 MHz, D₆ -DMSO) δ7.33 (1H, m), 7.51-7.54 (3H, m).

N-[3(R)-5-Cyclohexyl-2,3-dihydro-1-methyl-2-oxo-1H-1,4-benzodiazepin-3-yl]N'-[3-(5-oxo-4H-1,2,4-oxadiazolin,3-yl) phenyl]urea

A suspension of 1-(5-oxo-4H-1,2,4-oxadiazolin-3-yl)-3-aminobenzenehydrochloride salt [Intermediate 2] (281 mg, 1.3 mmol) in anhydroustetrahydrofuran (25 ml), under nitrogen, was treated with triethylamine(0.36 ml, 2.6rnmol) dropwise. The mixture was then cooled to 0° C. andtriphosgene (127 mg, 0.43 mmol) added, followed by triethylamine (0.36ml, 2.6 mmol) dropwise. The mixture was stirred at 0° C. for 5 min thenthe cooling bath removed and stirred at ambient temperature for 10 min.The mixture was then cooled to 0° C. and a solution of(+)-3R)-amino-5-cyclohexyl-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-3-one[Intermediate 1 ] (245 mg, 0.9 mmol) in anhydrous tetrahydrofuran (4 ml)added dropwise. After addition the suspension was stirred at 0° C. for 5min, then the cooling bath was removed and the mixture stirred at roomtemperature for 30 min. The mixture was then filtered and the filtrateevaporated in vacuo. The residue was partitioned between ethyl acetate(20 ml) and 20% aqueous acetic acid (20 ml). The organic layer wasseparated and washed once more with 20% aqueous acetic acid (20 ml)followed by brine (20ml). The organic phase was separated, dried (Na₂SO₄) and evaporated. The residue was chromatographed on silica gel,eluting with dichloromethane:methanol (97:3), to afford the desiredproduct as a viscous oil. This was azeotroped with dichloromethane (2×20ml) followed by toluene (2×20 ml). The resultant white solid was thentriturated with anhydrous ether to afford the desired urea (310 mg, 73%)as a white solid. mp 216-218° C. (petrol (60/80)/EtOAc). ¹ H NMR (360MHz, D₆ -DMSO) δ0.91 (1H, m), 1.09tl.91 (9H, m), 2.93 (1H, m), 3.32 (3H,s), 5.07 (1H, d, J=8 Hz), 7.32-7.44 (4H, m), 7.55 (2H, m), 7.64 (1H, dd,J=8 and 8 Hz), 7.75 (1H, d, J=8 Hz), 7.91 (1H, s), 9.25 (1H, s).

EXAMPLE 2N-[3(R,S)-5-Cyclobutyl-2,3-dihydro-1-methyl-2-oxo-1H-1,4-benzodiazepin-3-yl]N'-[3-(5-oxo-4H-1,2,4-oxadiazolin-3-yl)phenyl]urea

Step 1: 2-Aminophenyl cyclobutyl methanone

Over a period of 1 h a solution of cyclobutylbromide (13 g, 0.1 mmol) indiethyl ether (150 ml) was added dropwise to a slurry of magnesiumturnings (2.5 g, 0.11 mol) and a crystal of iodine in diethyl ether (20ml) at reflux. The mixture was stirred for a further hour whereupon theGrignard solution was cannulated into a pressure equalising droppingfunnel, attached to a three-necked round-bottomed flask, which was underan atmosphere of nitrogen.

A solution of aminobenzonitrile (3.78 g, 32 mmol) at 0° C. in diethylether (50 ml) was treated dropwise with the Grignard reagent preparedabove, over a period of 15 min.

Once the addition was complete, the mixture was warmed to roomtemperature and stirred for 16 h under nitrogen. The solution was cooledto 0° C., quenched with 5N hydrochloric acid (20 ml), and basified usingsolid sodium hydroxide (4 g). The aqueous solution was extracted withethyl acetate (2×100 ml) and the combined organic layers were dried (Na₂SO₄) and evaporated. The residue was chromtographed on silica gel using2:1 petrol: ethyl acetate as the eluant. This gave a yellow oil whichwas then azeotroped with toluene (2×80 ml) to give the title compound (4g, 71%) as a pale yellow solid. mp 55° C. ¹ H NMR (250 MHz,CDCl₃)δ1.72-2.48 (6H, m), 3.80-4.00 (1H, m), 6.23 (2H, brs), 6.50-6.61(2H, m), 7.11-7.22 (1H, m), 7.45-7.54 (1H m).

Step 2:3(R,S)-[(Benzyloxycarbonyl)amino]-5-cyclobutyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one

A solution of α-isopropylthio-N-benzyloxycarbonyl glycine (8.4 g, 29.7mmol) in anhydrous dichloromethane (200 ml) was cooled to 0° C.N-Methylmorpholine (3.3 ml, 29.7 mmol) was added over 2 min followed byisobutyl chloroformate (3.9 ml, 29.7 mmol). This mixture was stirred for15 min at 0° C. whereupon the mixture was heated to reflux.2-Aminophenyl cyclobutyl methanone (4 g, 22.9 mmol ) in anhydrousdichloromethane (20 ml) was added dropwise at reflux to the reactionmixture over 10 min and the mixture stirred at reflux for a further 1.5h. The reaction mixture was washed with 1N citric add (100 ml), water(100 ml), saturated sodium bicarbonate solution (100 ml) and brine (100ml). The organic phase was dried (Na₂ SO₄), evaporated and azeotropedwith toluene (2×100 ml) to give a yellow oil. Trituration with 7:1petrol:ethyl acetate afforded the product (8 g, 80%) as a colourlesssolid. This material was used without further purification.

A solution of anhydrous tetrahydrofuran (300 ml) was cooled to 0° C. andsaturated with ammonia gas. To this solution was added the glycinamide(8 g, 18 mmol) prepared above, followed by mercuric chloride (7.4 g, 27mmol). The mixture was stirred at 0° C. for 1.5 h with continuousbubbling of ammonia gas. The mixture was filtered through "hyflo" andthe filtrate evaporated to afford the desired amine as a colourless waxysolid. The material was used without further purification.

The amine (6.9 g, 18 mmol) prepared above was dissolved in acetic add(250 ml) and treated with ammonium acetate (6.5 g, 84.6 mmol). Thismixture was stirred at room temperature for 16 h under nitrogen. Thesolvent was evaporated and the residue partitioned between ethyl acetate(250 ml) and 10% sodium hydroxide solution (100 ml). The organic wasseparated, dried (Na₂ SO₄) and evaporated to give a yellow solid.Trituration with diethyl ether afforded the title compound (3.8 g, 50%)as a colourless solid. mp 200-202° C. TLC (silica, petrol:ethyl acetate2:1). Rf=0.3. ¹ H NMR (250 MHz, CDCl₃)δ1.60-2.80 (6H, m), 3.70 (1H, m),5.12 (2H, m), 5.22 (1H, d, J: 8 Hz), 6.50 (1H, d, J=8 Hz), 7.02-7.53(9H, m), 9.44 (1H, s).

Step 3: 3(R,S)-[(BenzyloxVcarbonyl)amino]-5-cyclobutyl-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-3-one

3(R,S)-[(Benzyloxycarbonyl)amino]-5-cyclobutyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one(1 g, 2.75 mmol) in anhydrous toluene (70 ml) was heated to reflux. Asolution of dimethylformamide dim ethyl acetal (1.75 ml, 13.7 mmol) inanhydrous toluene (10 ml) was added dropwise and the mixture was heatedat reflux for a further 3 h. The solvent was evaporated and the residuetriturated with diethyl ether to afford the tifie compound (0.75 g, 72%)as a colourless solid. mp 210-211° C. ¹ H NMR (250 MHz, CDCl₃)δ1.68-2.06(4H, m), 2.20-2.60 (2H, m), 3.41 (3H, s), 3.60-3.80 (1H, m), 5.00-5.30(3H, m), 6.51 (1H, d, J: 14 Hz), 7.14-7.54 (9H, m).

Step 4:3(R,S)-Amino-5-cyclobutyl-1,3-dihydro-1-methyl-2H-1,4-benzodiazenin-2-one

3(R,S)-[(Benzloxycarbonyl)amino]-5-cyclobutyl-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one(400 mg, 1.06 mmol) was treated with a solution of 45% hydrogen bromidein acetic add (10 ml), and stirred for 20 min at room temperature. Themixture was then added dropwise onto cold (0° C.) diethyl ether (50 ml).A white solid was precipitated and filtered off. The solid was treatedwith 10% sodium hydroxide solution (50 ml), then extracted with ethylacetate (80 ml). The organic layer was separated, dried (Na₂ SO₄) andevaporated to give a yellow foam. This material was then used withoutfurther purification.

Step 5:N-[3(R,S)-5-Cyclobutyl-2,3-dihydro-1-methyl-2-oxo-1H-1,4-benzodiazepin-3-yl]N'[3-(5-oxo-4H-1,2,4-oxadiazolin-3-yl)phenyl]urea

A suspension of 1-(5-oxo-4H-1,2,4-oxadiazolin-3-yl)-3-aminobenzenehydrochloride (330 mg, 1.54 mmol) in anhydrous tetrahydrofuran (25 ml)was treated with triethylamine (428 μl, 3.08 mmol) and stirred at roomtemperature for 5 min. The mixture was cooled to 0° C. whereupontriphosgene (151 mg, 0.51 mmol) was added. The mixture was stirred at 0°C. for 2 min, then triethylamine (428 μl, 3.08 mmol) was added dropwiseto adjust the solution to pH 9. The mixture was then stirred for afurther 5 min, allowed to warm to 15° C., and then re-cooled to 0° C.Then a solution of3(R,S)-amino-5-cyclobutyl-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one(251 mg, 1.06 mmol) in anhydrous tetrahydrofuran (4 ml) was addeddropwise over 5 min. The mixture was stirred at 0° C. for 5 min, allowedto warm to room temperature and then stirred for a further 40 min. Theundissolved material was removed by filtration. The solvent wasevaporated in vacuo and the residue partitioned between ethyl acetate(20 ml) and 20% aqueous acetic add (10 ml). The organic phase wasseparated, dried (Na₂ SO₄) and evaporated. The residue waschromatographed on silica gel with a 0.5% gradient elution of MeOH indichloromethane. Trituration with diethyl ether afforded the product(130 mg, 25%) as a colourless solid. mp 225° C. (dec.). ¹ H NMR (360MHz, D₆ -DMSO)δ1.72-1.87 (1H, m), 1.92-2.12 (3H, m), 2.27-2.42 (1H, m),2.46-2.59 (1H, m), 3.43 (3H, s), 3.72-3.84 (1H, m), 5.38 (1H, d, J=6Hz), 7.22-7.66 (8H, m), 7.70 (1H, d, J=8 Hz), 7.81 (1H, s), 9.03 (1H,s).

EXAMPLE 3N-[3(R,S)-5-Cyclohexyl-2,3-dihydro-1-methyl-2-oxo-1H-1,4-benzodiazepin-3-yl]N'-[3-(5-oxo-4H-1,2,4-oxadiazolin-3-yl)phenyl]urea

A suspension of 1-(5-oxo-4H-1,2,4-oxadiazolin-3-yl) -3-aminobenzenehydrochloride salt [Intermediate 2] (57 mg, 0.27 mmol) in anhydroustetrahydrofuran (5 ml), under nitrogen, was treated with triethylamine(75 μl, 0.55 mmol) dropwise. The mixture was then cooled to 0° C. andtriphosgene (26 mg, 0.09 mmol) added, followed by triethylamine (75 μl,0.55 mmol) dropwise. The mixture was then stirred at 0° C. for 5 minthen the cooling bath removed and stirred at ambient temperature for 10min. The mixture was then cooled to 0° C. and a solution of3(R,S)-amino-5-cyclohexyl-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-3-one[Intermediate 1, Step 5] in anhydrous tetrahydrofuran (3 ml) addeddropwise. After addition the suspension was stirred at 0° C. for 5 min,then the cooling bath was removed and the mixture stirred at roomtemperature for 30 min. The mixture was then filtered and the filtrateevaporated in vacuo. The residue was partitioned between ethyl acetate(20 ml) and 20% aqueous acetic acid (20 ml). The organic layer wasseparated and washed once more with 20% aqueous acetic acid (20 ml)followed by brine (20 ml). The organic phase was separated, dried (Na₂SO₄) and evaporated. The residue was treated withdichloromethane:methanol (97:3) and the undissolved solid filtered off.The white solid (35 mg, 41%) was identified as the desired urea. Thefiltrate was chromatographed on silica gel, eluting withdichloromethane:methanol (97:3), to afford the title compound (9 mg,10%) as a white solid. mp 197-199° C. ¹ H NMR data was as described forExample 1.

EXAMPLE 4A Tablets containing 1-25 mg of compound

    ______________________________________                   Amount mg    ______________________________________    Compound of formula (I)                      1.0       2.0   25.0    Microcrystalline cellulose                     20.0      20.0   20.0    Modified food corn starch                     20.0      20.0   20.0    Lactose          58.5      57.5   34.5    Magnesium Stearate                      0.5       0.5    0.5    ______________________________________

EXAMPLE 4B Tablets containing 26-100 mg of compound

    ______________________________________                   Amount mg    ______________________________________    Compound of formula (I)                     26.0      50.0    100.0    Microcrystalline cellulose                     80.0      80.0    80.0    Modified food corn starch                     80.0      80.0    80.0    Lactose          213.5     189.5   139.5    Magnesium Stearate                      0.5       0.5     0.5    ______________________________________

The compound of formula (I), cellulose, lactose and a portion of thecorn starch are mixed and granulated with 10% corn starch paste. Theresulting granulation is sieved, dried and blended with the remainder ofthe corn starch and the magnesium stearate. The resulting granulation isthen compressed into tablets containing 1.0 mg, 2.0 mg, 25.0 mg, 26.0mg, 50.0 mg and 100 mg of the active compound per tablet.

EXAMPLE 5 Parenteral injection

    ______________________________________                         Amount mg    ______________________________________    Compound of formula (I)                           1 to 100    Citric Acid Monohydrate                           0.75    Sodium Phosphate       4.5    Sodium Chloride        9    Water for Injections   to 1 ml    ______________________________________

The sodium phosphate, citric acid monohydrate and sodium chloride aredissolved in a portion of the water. The compound of formula (I) isdissolved or suspended in the solution and made up to volume.

EXAMPLE 6 Topical formulation

    ______________________________________                         Amount mg    ______________________________________    Compound of formula (I)                           1-10    Emulsifying Wax        30    Liquid paraffin        20    White Soft Paraffin    to 100    ______________________________________

The white soft paraffin is heated until molten. The liquid paraffin andemulsifying wax are incorporated and stirred until dissolved. Thecompound of formula (I) is added and stirring continued until dispersed.The mixture is then cooled until solid.

BIOLOGICAL ACTIVITY 1. CCK Receptor Binding (Pancreas)

CCK-8 sulphated was radiolabelled with ¹²⁵ I-Bolton Hunter reagent (2000Ci/mmole). Receptor binding was performed according to Chang and Lotti(Proc. Natl. Acad. Sci. 83, 4923-4926, 1986) with minor modifications.

Male Sprague-Dawley rats (150-200 g) were sacrificed by decapitation.The whole pancreas was dissected free of fat tissue and was homogenizedin 25 volumes of ice-cold 10 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulphonic acid (HEPES) buffer with 0.1% soya bean trypsin inhibitor (pH7.4 at 25° C.) with a Kinematica Polytron. The homogenates werecentrifuged at 47,800 g for 10 min. Pellets were resuspended in 10volumes of binding assay buffer (20mM (HEPES)), 1 mM ethyleneglycol-bis-(β-aminoethylether-N,N'-tetraacetic acid) (EGTA), 5 mM MgCl₂,150 mM NaCl, bacitracin 0.25 mg/ml, soya bean trypsin inhibitor 0.1mg/ml, and bovine serum albumin 2 mg/ml pH 6.5 at 25° C.) using a Teflon(trademark) homogenizer, 15 strokes at 500 rpm. The homogenate wasfurther diluted in binding assay buffer to give a final concentration of0.5 mg original wet weight/1 ml buffer. For the binding assay, 50 μl ofbuffer (for total binding) or unlabelled CCK-8 sulphated to give a finalconcentration of 1 μM (for nonspecific binding) or the compounds ofFormula I (for determination of inhibition of ¹²⁵ I-CCK-8 binding) and50 μl of 500 pM ¹²⁵ I-CCK-8 (i.e. 50 pM final concentration) were addedto 400 μl of the membrane suspensions in microfuge tubes. All assayswere run in duplicate. The reaction mixtures were incubated at 25° C.for 2 hours and the reaction terminated by rapid filtration (Brandell 24well cell harvester) over Whatman GF/C filters, washing 3×4 mls withice-cold 100 Mm NaCl. The radioactivity on the filters was counted witha LKB gamma counter.

2. CCK Receptor Binding (Brain)

CCK-8 sulphated was radiolabelled and the binding was performedaccording to the description for the pancreas method with minormodifications.

Male Hartley guinea pigs (300-500 g) were sacrificed by decapitation andthe cortex was removed and homogenized in 25 mL ice-cold 0.32 M sucrose.The homogenates were centrifuged at 1000 g for 10 minutes and theresulting supernatant was recentrifuged at 20,000 g for 20 minutes. TheP₂ pellet was resuspended in binding assay buffer (20 mM HEPES, 5 mMMgCl₂, 0.25 mg/ml bacitracin, 1 mM EGTA pH 6.5 at 25° C.), using aTeflon (trademark) homogenizer (5 strokes at 500 rpm) to give a finalconcentration of 10 mg original wet weight /1.2 ml buffer. For thebinding assay, 50 μl of buffer (for total binding) or unlabelled CCK-8sulphated to give a final concentration of 1 μM (for nonspecificbinding) or the compounds of Formula I (for determination of inhibitionof ¹²⁵ I-CCK-8 binding) and 50 μl of 500 pM ¹²⁵ I-CCK-8 (i.e. finalconcentration of 50 pM) were added to 400 μl of the membrane suspensionsin microfuge tubes. All assays were run in duplicate. The reactionmixtures were incubated at 25° C. for 2 hours and then the reaction wasterminated by rapid filtration (Brandell 24 well cell harvester) onWhatman GF/C filters with 3×5 ml washes of cold 100 mM NaCl. Theradioactivity on the filters was counted with a LKB gamma counter.

In Vitro Results Effects of the Compounds of Formula I on ²⁵ I-CCK-8Receptor Binding

The preferred compounds of Formula I are those which produceddose-dependent inhibition of specific ¹²⁵ I-CCK-8 binding as defined asthe difference between total and non-specific (i.e. in the presence of 1μM CCK) binding.

Drug displacement studies were performed with at least 10 concentrationsof compounds of Formula I and the IC₅₀ values were determined byregression analysis IC₅₀ refers to the concentration of the compoundrequired to inhibit 50% of specific binding of ¹²⁵ I-CCK-8.

The data in Table I were obtained for compounds of Formula I.

                  TABLE I    ______________________________________    CCK RECEPTOR BINDING RESULTS    IC.sub.50 (nM)    Compound       .sup.125 I-CCK                            .sup.125 I-CCK    of Ex #        Pancreas Brain    ______________________________________    1              500      0.123    2              110      5.98    ______________________________________

We claim:
 1. A compound of formula (I): ##STR12## wherein: R¹ representsH, (CH₂)_(q) imidazolyl, (CH₂)_(q) tetrazolyl, (CH₂)_(q) triazolyl,(where q is 1, 2 or 3); C₁₋₆ alkyl optionally substituted by one or moregroups selected from halo, hydroxy and NR⁶ R⁷ (where R⁶ and R⁷ eachindependently represents H or C₁₋₄ alkyl, or R⁶ and R⁷ together form achain (CH₂)_(p) where p is 4 or 5); C₃₋₇ cycloalkyl; cyclopropylmethyl;CH₂ CO₂ R⁵ (where R⁵ is C₁₋₄ alkyl), CH₂ CONR⁶ R⁷ or CH₂CH(OH)--W--(CH₂)₂ NR⁶ R⁷ where W is S or NH and R⁶ and R ⁷ are aspreviously defined;R² represents a group ##STR13## wherein: X representsO, S or NR⁸ where R⁸ represents H or C₁₋₄ alkyl; one of Z and Y is C═Oand the other is O, S or NR⁹, where R⁹ represents H or C₁₋₄ alkyl; R³represents C₁₋₆ alkyl, or halo; R⁴ represents C₃₋₇ cycloalkyl; and n is0, 1, or 2;or a pharmaceutically salt thereof.
 2. A compound as claimedin claim 1 selectedfrom:N-[3(R)-5-cyclohexyl-2,3-dihydro-1-methyl-2-oxo-1H-1,4-benzodiazepin-3-yl]N'-[3-(5-oxo-4H-1,2,4-oxadiazolin-3-yl)phenyl]urea;N-[3(R,S)-5-cyclobutyl-2,3-dihydro-1-methyl-2-oxo-1H-1,4-benzodiazepin-3-yl]N'-[3-(5-oxo-4H-1,2,4-oxadiazolin-3-yl)phenyl]urea;N-[3(R,S)-5-cyclohexyl-2,3-dihydro-1-methyl-2-oxo-1H-1,4-benzodiazepin-3-yl]N'-[3-(5-oxo-4H-1,2,4-oxadiazolin-3-yl)phenyl]urea;and pharmaceutically acceptable salts thereof.
 3. A compound as claimedin claim 1 wherein R¹ is C₁₋₆ alkyl.
 4. A compound as claimed in claim 1wherein R² is oxadiazolinone.